Neuroscience Center of Excellence, Department of Ophthalmology, Louisiana State University Health Sciences Center, New Orleans, Louisiana 70112, USA.
J Ocul Pharmacol Ther. 2011 Jun;27(3):235-41. doi: 10.1089/jop.2010.0187. Epub 2011 May 6.
The human corneal endothelium has a very low mitotic rate, and with aging there is a decrease in the number of cells. 15-epi-LXA4 is an anti-inflammatory, bioactive lipid formed when aspirin acetylates cyclooxygenease-2 and redirects cyclooxygenease-2 catalytic activity away from prostaglandins. The purpose of the current study was to evaluate the action of 15-epi-LXA4 in the endothelium viability of human corneas stored in Optisol-GS.
Human corneal endothelial (HCE) cells along with the Descemet's membrane were isolated from fresh human eyes obtained from National Disease Research Interchange. Cell phenotype was identified by using the tight junctions cell marker ZO-1. LXA4 receptor (FPR2/ALX) was detected by immunostaining of HCE cells and human corneal tissue using a polyclonal antibody. Cell proliferation was evaluated with Ki-67 antibody. To measure cell migration, confluent HCE cells were wounded by a linear scraping with a sterile pipette tip in the center of the well and incubated for 24 h with or without 15-epi-LXA4. To evaluate the reparative capacity of 15-epi-LXA4, 7 pairs of human corneas were incubated in Dulbecco's modified Eagle's medium/F12 media at 37°C with or without 100 nM 15-epi-LXA4 for 24 h and then stored at 4°C in Optisol-GS for 12 days. Endothelial viability was assessed by 2 staining techniques: a viability/cytotoxicity kit and trypan blue combined with alizarin red S.
HCE cells and the endothelium of human corneal sections strongly expressed the LXA4 receptor. There was a 3-fold increase in cell proliferation when HCE cells were incubated with 100 nM 15-epi-LXA4 for 24 h. No significant migration was observed after 24 h incubation with 15-epi-LXA4. Corneas incubated for 24 h in Dulbecco's modified Eagle's medium/F12 media in the presence of 15-epi-LXA4 and then stored for 12 days in Optisol-GS had a 36% to 56% increase in viability compared with controls without 15-epi-LXA4.
15-epi-LXA4 is an important mediator that protects the integrity of the human endothelium during corneal preservation in Optisol-GS.
人眼角膜内皮细胞有非常低的有丝分裂率，随着年龄的增长，细胞数量会减少。15-epi-LXA4 是一种抗炎、生物活性脂质，在阿司匹林乙酰化环氧化酶-2 并将环氧化酶-2 的催化活性从前列腺素转移时形成。本研究的目的是评估 15-epi-LXA4 在 Optisol-GS 中储存的人眼角膜内皮活力中的作用。
从国家疾病研究交流处获得的新鲜人眼中分离出人眼角膜内皮 (HCE) 细胞和 Descemet 膜。通过使用紧密连接细胞标记物 ZO-1 鉴定细胞表型。使用多克隆抗体通过免疫染色 HCE 细胞和人角膜组织来检测 LXA4 受体 (FPR2/ALX)。通过 Ki-67 抗体评估细胞增殖。为了测量细胞迁移，将汇合的 HCE 细胞用无菌移液管尖端在孔的中心线性划伤，并用或不用 15-epi-LXA4 孵育 24 小时。为了评估 15-epi-LXA4 的修复能力，将 7 对人角膜在 37°C 的 Dulbecco 修改的 Eagle 培养基/F12 培养基中孵育，用或不用 100 nM 15-epi-LXA4 24 小时，然后在 4°C 的 Optisol-GS 中储存 12 天。通过两种染色技术评估内皮细胞活力：活力/细胞毒性试剂盒和台盼蓝结合茜素红 S。
HCE 细胞和人角膜切片的内皮强烈表达 LXA4 受体。当 HCE 细胞在 100 nM 15-epi-LXA4 中孵育 24 小时时，细胞增殖增加了 3 倍。孵育 24 小时后，用 15-epi-LXA4 观察到的细胞迁移没有明显变化。在用 15-epi-LXA4 在 Dulbecco 修改的 Eagle 培养基/F12 培养基中孵育 24 小时然后在 Optisol-GS 中储存 12 天的角膜中，与没有 15-epi-LXA4 的对照相比，活力增加了 36%至 56%。
15-epi-LXA4 是一种重要的介质，可在 Optisol-GS 中保存角膜期间保护人内皮的完整性。
